摘要
目的观察转化生长因子β1(TGF-β1)对CCAAT增强子结合蛋白α(C/EBPα)、过氧化物酶增殖物激活受体γ(PPARγ)核转录因子表达的影响,探讨C/EBPα在TGF-β1致肾间质纤维化过程中的表达及其机制。方法体外培养人肾小管上皮细胞(HK-2),利用TGF-β1分别在不同质量浓度(5和10 ng/m L)和不同时间点(0、4、12、24 h)刺激HK-2细胞,收集细胞总RNA和蛋白(核蛋白和总蛋白)以及细胞上清液。应用realtime PCR法和Western blotting检测C/EBPα和核转录因子PPARγ以及上皮细胞-间充质转化(EMT)相关蛋白α-SMA、E-cadherin的表达变化;ELISA方法检测细胞上清液中单核细胞趋化蛋白1(MCP-1)的表达水平。结果 TGF-β1(5 ng/m L)刺激HK-2细胞24 h后C/EBPα蛋白与PPARγ变化一致,差异均无统计学意义(P>0.05);E-cadherin表达减少。TGF-β1(10 ng/m L)分别刺激0、4、12、24 h后,核蛋白中C/EBPα在12 h时表达明显降低,PPARγ在4 h表达升高。以TGF-β1 10 ng/m L刺激HK-2细胞12 h后,C/EBPα、PPARγ、E-cadherin mRNA分别降低70%、50%和90%(P<0.05)。以10 ng/m L TGF-β1刺激HK-2细胞后,MCP-1在12、24 h时分别升高6倍和10.67倍。结论 C/EBPα参与TGFβ1诱导的肾间质纤维化过程,并且与间质纤维化过程中的炎症反应和EMT过程相关;且在此过程中,与PPARγ相互作用,促进或抑制肾间质纤维化。
Objective To observe the effect of transforming growth factor β1（TGF-β1） on expressions of CCAAT enhancer binding protein α（C/EBPα） and nuclear transcription factors of peroxisome proliferatoractivated receptor γ（PPARγ） and explore the expression of C/EBPα in the process of TGF-β1 induced renal fibrosis and relevant mechanisms.Methods Human renal tubular epithelial cells（HK-2） were cultured in vitro and stimulated by TGF-β1 of different concentrations（5 and 10 ng/mL） at different time points（0,4,12,and24 h）.Total RNA and protein（nucleoprotein and total protein） and supernatant were collected.Changes of expressions of C/EBPα,nuclear transcription factor PPARγ,and epithelial-mesenchymal transition（EMT）related proteins α-SMA and E-cadherin were detected by real-time PCR and Western blotting.The expression level of monocyte chemotactic protein 1（MCP-1） in supernatant was detected by ELISA.Results The change of C/EBPα level was the same as that of PPARγ level after HK-2 cells were stimulated by TGF-β1（5 ng/mL）for 24 h.The difference was not statistically significant（P〉0.05） and the expression of E-cadherin decreased.The expression of C/EBPα in nucleoprotein decreased remarkably at 12 h and the expression of PPARγincreased at 4 h after HK-2 cells were stimulated by TGF-β1（10 ng/mL） for 0,4,12,and 24 h.The mRNA expressions of E-cadherin,C/EBPα,and PPARγ decreased by 70%,50%,and 90%,respectively after HK-2cells were stimulated by TGF-β1（10 ng/mL） for 12 h（P〉0.05）.The MCP-1 level increased by 6 and 10.67 times at 12 h and 24 h after HK-2 cells were stimulated by TGF-β1（10 ng/mL）.Conclusion C/EBPαinvolves in the renal interstitial fibrosis induced by TGF-β1 and is relevant to inflammatory response and EMT process of the renal interstitial fibrosis.C/EBPα also interacts with PPARγ and enhances or inhibits the renal interstitial fibrosis.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2015年第9期1243-1247,共5页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(30270613
30771000
81270782)
国家重点基础研究发展计划("973"计划)(2012CB517701
2012CB517604)
国家"十二五"科技攻关项目(2011BAI10B00)~~
